| 货号 | 7105S |
| 描述 | CST’s PathScan® Apoptosis Multi-Target Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that combines the reagents necessary to detect endogenous levels of p53 protein, phospho-p53 protein (Ser15), Bad, phospho-Bad (Ser112), Cleaved Caspase-3 (Asp175) and Cleaved PARP (Asp214). These molecules represent key signaling proteins in pathways controlling survival and apoptosis. Sixteen assays are provided for each target protein. Specific assay formulations for the indicated target proteins can be found in the datasheets associated with the individual sandwich ELISA kits*. Briefly, a capture antibody** has been coated onto the microwells. After incubation with cell lysates, the target protein is captured by the coated antibody. Following extensive washing, a detection antibody** is added to detect the captured target protein. An HRP-linked secondary antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of bound target protein. *See companion products. **Antibodies in kit are custom formulations specific to kit. |
| 反应种属 | Human/Monkey |
| 应用 | ELISA |
| 目标/特异性 | CSTs PathScan® Apoptosis Multi-Target Sandwich ELISA Kit #7105 detects endogenous levels of six proteins: total p53, phospho-p53 (Ser15), total Bad, phospho-Bad (Ser112), cleaved caspase-3 (Asp175) and cleaved PARP (Asp214). Activation of these proteins can be observed over time in response to toxic chemical compounds. As shown in Figures 1 and 2, both doxorubicin and staurosporine can induce apoptosis in HeLa cells, evidenced by increased levels of cleaved PARP and caspase-3. However, treatment with doxorubicin, which damages cellular DNA, induces p53 phosphorylation at Ser15 and stabilizes p53, while treatment with staurosporine, a kinase inhibitor, has no effect on p53 phosphorylation. While total Bad and phospho-Bad (Ser112) levels are relatively consistent after doxorubicin treatment, a gradual decline of both targets was observed after staurosporine treatment. COS cells are resistant to apoptosis due to high constitutive levels of p53. Therefore, the same dose of doxorubicin applied to HeLa cells only induces low amounts of apoptosis in these cells as evidenced by cleaved caspase-3 and cleaved PARP protein levels (Figure 3). The relationship between the protein concentration of the lysate and the absorbance at 450 nm can be found in the datasheets associated with the individual PathScan® Sandwich ELISA Kits*. *See companion products. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species. |
| 供应商 | CST |
| 背景 | Apoptosis is a regulated physiological process leading to cell death. Caspases, a family of cysteine acid proteases, are central regulators of apoptosis.Initiator caspases (including 8, 9, 10 and 12) are closely coupled to proapoptotic signals. Once activated, these caspases cleave and activate downstream effector caspases (including 3, 6 and 7), which in turn cleave cytoskeletal and nuclear proteins like PARP, α-fodrin, DFF and lamin A, and induce apoptosis.Cytochrome c released from mitochondria is coupled to the activation of caspase-9, a key initiator caspase (1). Proapoptotic stimuli include the FasL, TNF-α, DNA damage and ER stress. Fas and TNFR activate caspases 8 and 10 (2), DNA damage leads to the activation of caspase-9 and ER stress leads to the calcium-mediated activation of caspase-12 (3). The inhibitor of apoptosis protein (IAP) family includes XIAP and survivin and functions by binding and inhibiting several caspases (4,5). Smac/Diablo, a mitochondrial protein, is released into the cytosol upon mitochondrial stress and competes with caspases for binding of IAPs. The interaction of Smac/Diablo with IAPs relieves the inhibitory effects of the IAPs on caspases (6). |
| 存放说明 | 4C |
| 参考文献 | Baker, S.J. and Reddy, E.P. (1998) Oncogene 17, 3261-3270. Budihardjo, I. et al. (1999) Annu. Rev. Cell Dev. Biol. 15, 269-290. Nakagawa, T. et al. (2000) Nature 403, 98-103. Deveraux, Q. L. et al. (1998) EMBO J. 17, 2215-2223. Li, F. et al. (1998) Nature 396, 580-584. Du, C. et al. (2000) Cell 102, 33-42. |
Figure 1. Treatment of HeLa cells with doxorubicin induces phosphorylation of p53 at Ser15, as well as cleavage of PARP and caspase-3 as detected by PathScan® Apoptosis Multi-Target Sandwich ELISA Kit #7105 and Western analysis. HeLa cells (80-90% confluent) were starved overnight and stimulated with doxorubicin (5 μM at 37ºC for indicated times). Lysates were assayed at a protein concentration of 1 mg/ml. The absorbance readings at 450 nm are shown as a 3-dimensional representation in the left panel, while the corresponding Western blots are shown in the right panel. Antibodies used for Western analysis include Phospho-p53 (Ser15) Antibody #9284, p53 Antibody #9282, Cleaved Caspase-3 (Asp175) Antibody #9661 and PARP Antibody #9542. Total Bad and phospho-Bad (Ser112) proteins were not detected by Western due to low endogenous levels in HeLa cells. 图1. 使用PathScan® Apoptosis Multi-Target Sandwich ELISA Kit #7105和Western，分析经doxorubicin诱导p53 Ser15磷酸化，或者PARP和caspase-3裂解的HeLa细胞。HeLa细胞（铺满80％-90％）饥饿过夜，doxorubicin刺激（5 μM， 37℃，指定时间）。蛋白质浓度为1 mg/ml 时测定裂解液。在450 nm处的吸光度读数，显示为一个3维图见左图，而相应的Western blots结果显示在右图。用于Western分析的抗体包括Phospho-p53 (Ser15) Antibody #9284、p53 Antibody #9282、Cleaved Caspase-3 (Asp175) Antibody #9661和PARP Antibody #9542。由于在HeLa细胞中的低内源性水平，Western未检出总Bad和磷酸化Bad (Ser112)蛋白的表达。 | |
Figure 2. Treatment of HeLa cells with staurosporine induces cleavage of PARP and caspase-3 but not phosphorylation of p53 at Ser15 as detected by PathScan® Apoptosis Multi-Target Sandwich ELISA Kit #7105 and Western analysis. HeLa cells (80-90% confluent) were starved overnight and stimulated with staurosporine (2 μM at 37ºC for indicated times). Lysates were assayed at a protein concentration of 1 mg/ml. The absorbance readings at 450 nm are shown as a 3-dimensional representation in the left panel, while the corresponding Western blots are shown in the right panel. The antibodies used for the Western analyses include Phospho-p53 (Ser15) Antibody #9284, p53 Antibody #9282, Cleaved Caspase-3 (Asp175) Antibody #9661 and PARP Antibody #9542. Total Bad and Phospho-Bad (Ser112) proteins were not detected by Western due to low endogenous levels in HeLa cells. 图2. 使用PathScan® Apoptosis Multi-Target Sandwich ELISA Kit #7105和Western分析星形孢菌素处理HeLa细胞诱导的PARP和caspase-3裂解，但是未有p53 Ser15磷酸化。HeLa细胞（80％-90％铺满）饥饿过夜培养，星形孢菌素刺激（2 μM，于37℃，指定时间）。蛋白质浓度为1 mg/ml 时测定裂解液。在450 nm处的吸光度读数显示为左图一个3维图，而相应的Western blots结果显示在右图。用于Western分析的抗体包括Phospho-p53 (Ser15) Antibody #9284、p53 Antibody #9282、Cleaved Caspase-3 (Asp175) Antibody #9661和PARP Antibody #9542。由于在HeLa细胞中的内源性水平低，总Bad和磷酸化Bad (Ser112)蛋白未被Western检出。 | |
